The integrity of the genome is of prime importance to a dividing cell. In response to DNA damage, eukaryotic cells rely upon a complex system of checkpoint controls to delay cell-cycle progression. The normal eukaryotic cell-cycle is divided into 4 phases (sequentially G1, S, G2, M) which correlate with distinct cell morphology and biochemical activity, and cells withdrawn from the cell-cycle are said to be in G0, or non-cycling state. When cells within the cell-cycle are actively replicating, duplication of DNA occurs in the S phase, and active division of the cell occurs in M phase. See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 36, 1997). DNA is organized in the eukaryotic cell into successively higher levels of organization that result in the formation of chromosomes. Non-sex chromosomes are normally present in pairs, and during cell division, the DNA of each chromosome replicates resulting in paired chromatids. (See generally Benjamin Lewin, GENES VI (Oxford University Press, Oxford, GB, Chapter 5, 1997).
Checkpoint delays provide time for repair of damaged DNA prior to its replication in S-phase and prior to segregation of chromatids in M-phase (Hartwell and Weinert, 1989, Science 246: 629-634). In many cases the DNA-damage response pathways cause arrest by inhibiting the activity of the cyclin-dependent kinases (Elledge, 1997, Science, 274: 1664-1671). In human cells the DNA-damage induced G2 delay is largely dependent on inhibitory phosphorylation of Cdc2 (Blasina et al., 1997, Mol. Cell Biol., 8: 1-11; Jin et al., 1996, J. Cell Biol., 134: 963-970), and is therefore likely to result from a change in the activity of the opposing kinases and phosphatases that act on Cdc2. However, evidence that the activity of these enzymes is substantially altered in response to DNA damage is lacking (Poon et al., 1997, Cancer Res., 57: 5168-5178).
Three distinct Cdc25 proteins are expressed in human cells. Cdc25A is specifically required for the G1-S transition (Hoffmann et al., 1994, EMBO J., 13: 4302-4310; Jinno et al., 1994, EMBO J. 13: 1549-1556), whereas Cdc25B and Cdc25C are required for the G2-M transition (Gabrielli et al., 1996, J. Cell Sci., 7: 1081-1093; Galaktionov et al., 1991, Cell, 67: 1181-1194; Millar et al., 1991, Proc. Natl. Acad. Sci. USA, 88: 10500-10504; Nishijima et al., 1997, J. Cell Biol., 138: 1105-1116). The exact contribution of Cdc25B and Cdc25C to M-phase progression is not known.
Much of our current knowledge about checkpoint control has been obtained from studies using budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast. A number of reviews of our current understanding of cell cycle checkpoints in yeast and higher eukaryotes have recently been published (Hartwell and Kastan, 1994, Science 266: 1821-1828; Murray, 1994, Current Biology, 6: 872-876; Elledge, 1996, Science, 274: 1664-1672; Kaufmann and Paules, 1996, FASEB J., 10: 238-247). In the fission yeast six gene products, rad1+, rad3+, rad9+, rad17+, rad26+, and hus1+ have been identified as components of both the DNA-damage dependent and DNA-replication dependent checkpoint pathways. In addition cds1+ has been identified as being required for the DNA-replication dependent checkpoint and rad27+/chk1+ has been identified as required for the DNA-damage dependent checkpoint in yeast.
Several of these genes have structural homologues in the budding yeast and further conservation across eukaryotes has recently been suggested with the cloning of two human homologues of S. pombe rad3+: ATM (ataxia telangiectasia mutated) (Savitsky et al., 1995, Science, 268: 1749-1753) and ATR (ataxia telangiectasia and rad3+ related)(Bentley et al, 1996, EMBO J., 15: 6641-6651; Cimprich et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 2850-2855) and of a human homologue of S. pombe rad9+ (Lieberman et al., 1996, Proc. Natl. Acad. Sci. USA, 93: 13890-13885).
While much is known about yeast checkpoint proteins and genes, this knowledge is not fully predictive of the existence of corresponding human genes or proteins, or their effector role in human cell-cycle control and regulation.
In order to develop new and more effective treatments and therapeutics for the amelioration of the effects of cancer, it is important to identify and characterize human checkpoint proteins and to identify mediators of their activity.
The present invention is directed to the discovery of a novel human checkpoint kinase gene hCDS1, protein and constructs and methods for the production and use of hCDS1.
In particular, the present invention encompasses a nucleic acid sequence which encodes for hCDS1, consisting of the nucleic acid sequence of SEQ ID NO.: 1. In particular, the invention encompasses the nucleic acid sequence from position 66 to 1694 of the nucleic acid sequence of SEQ ID NO.: 1, which translates into the hCDS1 protein. The present invention also encompasses nucleic acid constructs, vectors, plasmids, cosmids and the like which contain the nucleic acid sequence of SEQ ID NO.: 1. In particular, the present invention provides for nucleic acid vector constructs which contain the nucleic acid sequence of SEQ ID NO.: 1 and are capable of expressing protein from this nucleic acid sequence. The present invention encompasses nucleic acid vectors that are suitable for the transformation of host cells, whether eukaryotic or prokaryotic, suitable for incorporation into viral vectors, or suitable for in vitro protein expression. The present invention further embodies the nucleic acid sequence of SEQ ID NO.: 1 in tandem with, or otherwise in conjunction with additional nucleic acids for the generation of fusion protein products containing at least the functional segment of the protein encoded for by the nucleic acid of SEQ ID NO.: 1. The present invention also encompasses the nucleic acid of SEQ ID NO.: 1 adapted for use as a naked DNA transformant for incorporation and expression in target cells. The present invention also provides for anti-sense DNA molecule formulations which are the complement to nucleic acid sequence of SEQ ID NO.: 1, and fragments thereof, whether complementary to contiguous or discontinuous portions of the nucleic acid sequence of SEQ ID NO.: 1. The present invention also provides for compositions incorporating modified nucleotides or backbone components which encode for the nucleic acid sequence of SEQ ID NO.: 1, its complement, or fragments thereof. Such modified nucleotides and nucleic acids are known in the art (see for example Verma et al., Ann. Rev. Biochem. 67: 99-134 (1998)). Thus the present invention encompasses modified nucleic acids which incorporate, for example, intemucleotide linkage modification, base modifications, sugar modification, nonradioactive labels, nucleic acid cross-linking, and altered backbones including PNAs (polypeptide nucleic acids).
The present invention provides for the novel human checkpoint kinase protein hCDS1, which consists of the amino acid sequence of SEQ ID NO.: 2. The invention encompasses hCDS1 protein produced by recombinant DNA technology and expressed in vivo or in vitro. The invention thus encompasses hCDS1 protein produced by transformed host cells in small-scale or large-scale production. The invention encompasses complete hCDS1 protein, in either glycosylated or unglycosylated forms, produced by either eukaryotic or prokaryotic cells. The present invention provides for hCDS1 protein expressed from mammalian, insect, plant, bacterial, fungal, or any other suitable host cell. The present invention encompasses hCDS1 protein that is produced as a fusion protein product, conjugated to a solid support, or hCDS1 protein which is labeled with any chemical, radioactive, fluorescent, chemiluminescent or otherwise detectable marker. The present invention also provides for hCDS1 protein isolated from natural sources and enriched in purity over that found in nature. The present invention also provides for pharmaceutical formulations of hCDS1 protein and formulations of the hCDS1 protein in pharmaceutically acceptable carriers or excipients.
The present invention encompasses any nucleic acid sequence which would encode for the amino acid sequence of SEQ ID NO.: 2, and the embodiments of these nucleic acid sequences as described for SEQ ID NO.: 1, as the nucleic acid code for generating any nucleic acid sequence which will encode for a protein having the amino acid sequence of SEQ ID NO.: 2 is predictable to one of skill in the art.
The present invention encompasses antibodies which bind specifically to the hCDS1 protein, either polyclonal or monoclonal, as generated by the immunization of a mammal with protein having the amino acid sequence of SEQ ID NO.: 2, or fragments thereof.
The present invention also encompasses equivalent proteins where substitutions of amino acids in the sequence of SEQ ID NO.: 2 that are reasonably predictable as being equivalent, and the embodiments thereof as described for SEQ ID NO.: 2. For example, non-polar (hydrophobic side-chain) amino acids alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine; uncharged polar amino acids glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine; charged polar amino acids aspartic acid, glutamic acid; basic amino acids lysine, arginine, and histidine are understood by those in the art to have functionally predictable effects when substituted. Thus the present invention also encompasses equivalent nucleic acids which encode for such equivalent proteins and the embodinients thereof as described for SEQ ID NO.: 1.
The invention also provides for methods of generating hCDS1 protein, by using recombinant DNA technology and the appropriate nucleic acid encoding for hCDS1 protein, fusion protein, or fragments thereof. The invention provides for incorporating an appropriate nucleic acid sequence into a suitable expression vector, the incorporation of any suitable control elements such as promoter, enhancer, either inducible or constitutively expressed. The invention provides for the use of expression vectors with or without at least one additional selectable marker or expressible protein. The invention provides for methods wherein a suitably constructed expression vector is transformed or otherwise introduced into a suitable host cell, and protein is expressed by such a host cell. Thus the present invention also provides for the transformed host cells, which are capable of producing hCDS1 protein, fusion protein, or fragments thereof.
The discovery that hCDS1 acts in coordination with Cdc25 in the DNA damage checkpoint allows for the use of the compounds of the invention in methods for therapeutic treatment of diseases which involve abnormal DNA damage checkpoint function. The present invention further provides for the use of the compounds of the present invention as therapeutics for the treatment of cancer. In particular, the present invention allows for the specific modification of the hCDS1 -Cdc25 DNA damage checkpoint in cells.
The present invention also encompasses methods for screening test compounds for efficacy in effecting the hCDS1 mediated checkpoint function of eukaryotic cells, said method comprising contacting a test compound to eukaryotic cells, and detecting any change in hCDS1 expression or function. Thus the invention further encompasses the method of screening wherein said detection of change in hCDS1 expression or function is accomplished by assaying for hCDS1 mRNA production, or by assaying for hCDS1 protein expression. In particular, the present invention allows for the screening of candidate substances for efficacy in modifying the DNA damage checkpoint by screening for any change in Cdc25 phosphorylation, or kinase activity. The compounds or substances identified by the assays of the invention, or compounds corresponding to such compounds or substances, can be used for the manufacture of pharmaceutical therapeutics.
Thus, in one embodiment the present invention provides for pharmaceutical compositions which include the hCDS1 protein, hCDS1 nucleic acid, hCDS1 anti-sense nucleic acids. In another embodiment, the present invention provides for compounds or substances identified as suitable for use as a therapeutic by the assays of the invention, in pharmaceutical formulations. These pharmaceutical compositions can further include chemotherapeutic agents for the use in treating cancer, or be administered in a regimen coordinated with the administration of other anti-cancer therapies. The present invention, in one embodiment thus encompasses methods for combined chemotherapy using the hCDS1 derived pharmaceuticals independently, and in combination with other chemotherapeutic agents, and in a second embodiment as admixtures with other anti-cancer therapeutics for single dose administration.
According to one aspect of the present invention, there is provided a nucleic acid encoding hCDS1 protein having the amino acid sequence illustrated in FIG. 2 (SEQ ID NO.: 2), or encoding a functional equivalent fragment, or bioprecursor of said protein. Preferably, the nucleic acid may be a DNA molecule such as a genomic DNA molecule and even more preferably a cDNA molecule, however it may also be RNA.
In a preferred embodiment, a nucleic acid encoding hCDS1 protein comprises the nucleic acid sequence represented by position 66 to 1694 of the sequence illustrated in FIG. 1 (SEQ ID NO.: 1), the complement thereof, or a nucleic acid sequence capable of hybridizing to either under high stringency conditions.
The nucleic acid sequences defined herein may, advantageously, be capable of hybridizing under low stringency conditions to nucleic acid sequences derived from family members to identify homologs therefrom or alternatively to identify nucleic acid sequences from other species.
As would be well known to those skilled in the art due to the degeneracy of the genetic code the nucleic acid sequences according to the invention may include substitutions therein yet which still encode the same amino acid sequence.
Advantageously, the nucleic acids according to the invention may be incorporated into an expression vector and may be subsequently used to transforrn, transfect or infect a suitable host cell. In such an expression vector the nucleic acid according to the invention is operably linked to a control sequence, such as a suitable promoter or the like, ensuring expression of the proteins according to the invention in a suitable host cell. The expression vector may, advantageously be a plasmid, cosmid, virus or other suitable vector. The expression vector and the host cell transfected, transformed or infected with the vector also form part of the present invention. Preferably, the host cell is a eukaryotic cell or a bacterial cell and even more preferably a mammalian cell or insect cell. Mammalian host cells are particularly advantageous because they provide the necessary post-translational modifications to the expressed proteins according to the invention, such as glycosylation or the like, which modifications confer optimal biological activity on said proteins, which when isolated may advantageously be used in diagnostic kits or the like.
The expression vector including said nucleic acid according to the invention may advantageously be used in vivo, such as in, for example, gene therapy.
According to a further aspect of the invention there is also provided a transgenic cell, tissue or organism comprising a transgene capable of expressing hCDS1 protein, which protein comprises the amino acid sequence illustrated in FIG. 2 (SEQ ID NO.: 2), or the amino acid sequence of a functional equivalent or bioprecursor or fragment therefor. The term xe2x80x9ctransgene capable of expressionxe2x80x9d as used herein means a suitable nucleic acid sequence which leads to expression of hCDS1 or proteins, having the same fumction and/or activity. The transgene may include, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromosomal state. Preferably, the transgene comprises the nucleic acid sequence encoding the proteins according to the invention as described herein, or a functional fragment of said nucleic acid. A functional fragment of said nucleic acid should be taken to mean a fragment of the gene comprising said nucleic acid coding for the proteins according to the invention or a functional equivalent, derivative or a non-functional derivative such as a dominant negative mutant, or bioprecursor of said proteins. For example, it would be readily apparent to persons skilled in the art that nucleotide substitutions or deletions may be used using routine techniques, which do not affect the protein sequence encoded by said nucleic acid, or which encode a functional protein according to the invention.
The hCDS1 protein expressed by said transgenic cell, tissue or organism or a functional equivalent or bioprecursor of said protein also forms part of the present invention.
Further provided by the present invention is an antisense molecule which is capable of hybridizing to the nucleic acid according to the invention. Advantageously, the antisense molecule according to the invention may be used as a medicament, or in the preparation of a medicament for the treatment of cancer and other proliferative diseases.
The present invention also advantageously provides nucleic acid sequences of at least approximately 15 nucleotides of a nucleic acid according to the invention and preferably from 15 to 50 nucleotides. These sequences may advantageously be used as probes or primers to initiate replication, or the like. Such nucleic acid sequences may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with the sample under hybridizing conditions and detecting for the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample.
Advantageously, the nucleic acid sequences, according to the invention may be produced using such recombinant or synthetic means, such as for example using PCR cloning mechanisms which generally involve making a pair of primers, which may be from approximately 15 to 50 nucleotides spanning a region of the gene which is desired to be cloned, bringing the primers into contact with mRNA, cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region (and where necessary first performing a reverse transcription step), isolating the amplified region or fragment and recovering the amplified DNA. Generally, such techniques as defined herein are well known in the art, such as described in Sambrook et. al., (Molecular Cloninz; a Laboratory Manual, 1989). Advantageously, human allelic variants of the nucleic acid according to the invention may be obtained by for example, probing genomic DNA libraries from a range of individuals for example from different populations, and other genotyping techniques. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients, using techniques well known in the art, for example, the Sanger dideoxy chain termination method, which may advantageously ascertain any predisposition of a patient to certain proliferative disorders.
Further provided by the present invention are isolated proteins having the amino acid sequences as illustrated in FIG. 2 (SEQ ID NO.: 2) or the amino acid sequence of a functional equivalent functional fragment or bioprecursor of said protein in addition to antibodies, monoclonal or polyclonal capable of binding to the amino acid sequences of these proteins or fragments thereof. As would be well known to those skilled in the art, the proteins according to the invention may comprise conservative substitutions, deletions or insertions wherein the protein comprises different amino acids than those disclosed in FIG. 2, yet which substitutions, deletions or insertions do not affect the activity of the proteins according to the invention or their ability to interact in the human cell cycle checkpoint pathway.
Preferred fragments include those comprising an epitope of the proteins according to the invention. The epitopes may be determined using, for example, peptide scanning techniques as described in Geysen et. al., Mol. Immunol., 23; 709-715 (1986).
The antibodies according to the invention may be produced according to techniques which are known to those skilled in the art. Monoclonal antibodies may be prepared using conventional hybridoma technology as described in Kohler F and Milstein C (1985), Nature 256, 495-497. Polyclonal antibodies may also be prepared using conventional technology well known to those skilled in the art, and which comprises inoculating a host animal, such as a mouse, with a protein or epitope according to the invention and recovering the immune serum. The present invention also includes fragments of whole antibodies which maintain their binding activity, such as for example, Fv, F(abxe2x80x2) and F(abxe2x80x2)2 fragments as well as single chain antibodies.
Advantageously, the nucleic acid and/or the proteins according to the invention may be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The pharmaceutical composition containing said nucleic acids according to the invention may, for example, be used in gene therapy. Such nucleic acids, according to the invention, may be administered naked, or packaged in protein capsules, lipid capsules, liposomes, membrane based capsules, virus protein, whole virus, cell vectors, bacterial cell hosts, altered mammalian cell hosts, or such suitable means for administration.
There is further provided by the present invention a method for detecting for the presence or absence of a nucleic acid according to the invention, in a biological sample, which method comprises, a) bringing said sample into contact with a probe comprising a nucleic acid or probe according to the invention under hybridizing conditions, and b) detecting for the presence of hybridization, for example, by the presence of any duplex or triplex formation between said probe and any nucleic acid present in said sample. Proteins according to the invention may also be detected by a) contacting said sample with an antibody to an epitope of a protein according to the invention under conditions which allow for the formation of an antibody-antigen complex, b) monitoring for the presence of any antigen-antibody complex.
Kits for detecting said nucleic acids and proteins are also provided by the present invention. A kit for detecting for the presence of a nucleic acid according to the invention in a biological sample may comprise (a) means for contacting the sample with a probe comprising a nucleic acid or a probe according to the invention and means for detecting for the presence of any duplex or triplex formation between said probe and any nucleic acid present in the sample.
Likewise, a kit for detecting for the presence of a protein according to the invention in a biological sample may comprise (a) means for contacting said sample with an antibody to an epitope of a protein according to the invention under conditions which allow for the formation of an antibody protein complex, and means for monitoring said sample for the presence of any protein antibody complex.
A further aspect of the present invention provides a method of determining whether a compound is an inhibitor or an activator of expression or activity of the proteins of the human cell cycle checkpoint pathway which method comprises contacting a cell expressing the proteins in said pathway with said compound and comparing the level of expression of any of the proteins of the checkpoint pathway of said cell against a cell which has not been contacted with said compound. Any compounds identified may then advantageously be used as a medicament or in the preparation of a medicament for treating cancer or proliferative disorders. Alternatively, the compounds may be included in a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent or excipient therefor. Advantageously, any compounds identified as an inhibitor of the cell checkpoint pathway may be included in a pharmaceutical composition according to the invention together with a cytotoxic agent, such as a DNA damaging chemotherapeutic agent, and a pharmaceutically acceptable carrier diluent or excipient therefor. Thus, the human cell cycle checkpoint inhibitor may enhance the chemotherapeutic effect of cytotoxic agents used in, for example, anti-cancer therapy.
There is also provided by the present invention a method for screening candidate substances for anti-cancer therapy, which method comprises a) providing a protein according to the present invention exhibiting kinase activity together with a substrate for said protein under conditions such that the kinase will act upon the substrate, b) bringing the protein and substrate into contact with a candidate substance, c) measuring the degree of any increase or decrease in the kinase activity of the protein, d) selecting a candidate substance which provides a decrease or increase in activity. Such a candidate substance may also be used as a medicament, or in the preparation of a medicament for the treatment of cancer or other such proliferative cell disorders.
The present invention also comprises a method of identifying other proteins active in the cell checkpoint pathway, which method comprises a) contacting a cell extract with an antibody to an epitope of a protein according to the invention, under appropriate binding conditions, b) identifying any antibody-protein complex and c) analyzing the complex to identify any protein bound to the antibody or protein which is other than the protein according to the invention.
Another method for identifying proteins involved in the cell checkpoint pathway utilizes a two-hybrid system developed in yeast by Chien et. al., supra (1991). This technique is based on functional in vivo reconstitution of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or the activating domain of the transcription factor, expressing in the host cell at least one second hybrid DNA sequence encoding putative binding proteins to be investigated together with the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion; detecting any binding of the protein being investigated with a protein according to the invention by detecting for the production of any reporter gene product in the host cell; optionally isolating second hybrid DNA sequences encoding the binding protein. In one embodiment of this aspect of the invention the method may comprise:
(a) constructing at least two nucleotide vectors, the first of which comprises a nucleotide segment encoding for a DNA binding domain of GAL4 protein operably linked to a nucleic acid sequence encoding a protein according to the present invention, the second vector comprising a nucleotide sequence encoding a protein binding domain of GAL4 operably linked to a nucleotide sequence encoding a protein to be tested,
(b) co-transforming each of said vectors into a yeast cell being deficient for transcription of genes encoding galactose metabolizing proteins, wherein interaction between said test protein and the protein according to the invention leads to transcription of galactose metabolic genes.